Abstract
Purpose
Burkitt lymphoma (BL) is a B-cell lymphoma frequently diagnosed in children. It is characterized by MYC translocations, which lead to the constitutive expression of the MYC oncogene. MYC contributes to miR-29 repression through an E-box MYC binding site on the miR-29b-1/miR-29a promoter region. We evaluated the role of miR-29a/b/c and their predicted targets in BL pathogenesis.
Methods
Mature sequences of miR-29a/b/c were transfected to the BL cell lines BL41 and Raji, and evaluated for DNMT3B, MCL1, BIM, CDK6, AKT and TCL1 protein expression as well as for MCL-1 and CDK6 mRNA expression. BL cells were treated with 5-aza-2′-deoxycytidine (decitabine) and evaluated for miR29 expressions and methylation status. DNMT3B inhibition was performed by DNMT3B siRNA.
Results
Ectopic expression of miR-29s in BL cells decreased CDK6, DNMT3B, TCL1 and MCL-1 protein levels, but CDK6 and MCL-1 mRNA expression was unaffected by miR-29. Decitabine enhanced miR-29 expression levels and decreased CDK6 protein expression. Additionally, inhibition of DNMT3B by siRNA increased miR-29a/b expression. Notably, the miR-29a/b1 and miR-29b2/c promoter genes showed methylated CpG sequences that were demethylated after decitabine treatments. Furthermore, MYC-negative tumours had higher levels of miR-29 expression compared with MYC-translocated cases, suggesting that MYC regulates miR-29 in BL tumours.
Conclusions
Our results suggest a significant role for miR-29s in BL pathogenesis in altering the expression of targets involved in critical cancer pathways, such as cell cycle control, apoptosis inhibition and DNA methylation. Moreover, methylation-mediated miR-29 epigenetic silencing may occur during BL development.
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Acknowledgements
We are grateful to all clinicians who followed the patients included in the study and Dr. Lidia Maria Magalhães de Rezende for her collaboration in the histopathological review. This work was supported by grants from INCT para Controle do Câncer: CNPq 573806/2008-0/FAPERJ E26/170.026/2008; Programa de Oncobiologia/Fundação do Câncer and FAPERJ E-26/110.375/2014; SWISS-BRIDGE Foundation, sub-project 1B/2014. LM and MCR were supported by grants from Ministério da Saúde/INCA.
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432_2017_2575_MOESM1_ESM.tif
Effects of miR-29 transient transfections on BL cell viability. Transfection efficiency was evaluated at 8 h and 24 h by qRT-PCR, 2−ΔΔCT method was used to calculate relative expression (a). Cell viability was evaluated in BL 41 and Raji cells 8 h and 24 h after transfection with the Trypan Blue exclusion assay (b). The data represent the median of three independent experiments and the corresponding standard error, * p<0.05; ** p<0.01; *** p<0.001 (TIF 10700 KB)
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Mazzoccoli, L., Robaina, M.C., Apa, A.G. et al. MiR-29 silencing modulates the expression of target genes related to proliferation, apoptosis and methylation in Burkitt lymphoma cells. J Cancer Res Clin Oncol 144, 483–497 (2018). https://doi.org/10.1007/s00432-017-2575-3
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DOI: https://doi.org/10.1007/s00432-017-2575-3